There is a strong demand for higher sensitivity in SPR (surface plasmon resonance) assays e.g. in concentration analysis, host cell protein assays, measurements of protein A leakage from columns and for the detection of a wide range of molecules and biomarkers in complex biological media including immunogenicity assays. In primary antibody screening a higher sensitivity and higher throughput is needed in SPR assays. An improvement of sensitivity could allow shorter injection times and by that means higher through-put.
One commonly used approach to increase sensitivity is to sequentially inject one enhancement reagent i.e. a polyclonal antibody after the injection of analyte. This will give a limited enhancement since there is a limited number of binding sites for the enhancement molecules. Pei, Wang and Wang enhanced the response of a biotinylated enhancement reagent by injection of a strepavidin-biotinylated protein complex in a SPR-instrument. However large complexes tend to lose their binding capacity to sensor chips provided with a dextran matrix. Pei, Wang and Wang used a flat CO2H layer (C1 sensor chip) in their study with large complexes but this chip has limitations in terms of unspecific binding and sensitivity.
Instead of injecting a large complex for enhancement one could build the large complex on the surface. Laminar flow-assisted dendritic amplification is a signal amplification method for biomolecular binding events in microchannels (Hosokawa et al 2007) and JP 2008292270. However, the solution relies on presenting two combinable substances in parallel laminar flows which combines in a boundary layer in a narrowly defined detection zone. This creates high demands on the flow conditions and limits also the choices of detection device.
One could achieve a dendritic amplification by alternately inject the two enhancement molecules with ordinary injections (Yamaguchi and Harada). However repeated injections of two enhancement molecules is time consuming and also consumes considerable amounts of material.
All the above described prior art methods present significant limitations for efficient and high enhancement amplifications in flow cell assays formats, such as lengthy assays with multiple steps, high consumption of enhancement reagents, demands on delicate detection configurations as well as unspecific binding and lack of sensitivity. There is therefore a need for a quick an easy-to-perform assay that permits determination of an analyte by using an amplification step that is well suited for flow cell applications.